Dynamic changes in endoglin expression in the developing mouse heart

Endoglin (ENG) is essential for cardiovascular development and is expressed in the heart from its earliest developmental stages. ENG expression has been reported in the cardiac crescent, endocardium, valve mesenchyme and coronary vascular endothelial cells. However, its expression in these cell types is non-uniform and the dynamic changes in ENG expression during heart development have not been systematically studied.Using immunofluorescent staining we tracked ENG protein expression in mouse embryonic hearts aged from 11.5 to 17.5 days, and in postnatal and adult hearts. ENG is expressed in the endocardium and in venous endothelial cells throughout these developmental stages. ENG protein is down-regulated by approximately two-fold as a subset of early coronary veins reprogram to form arteries within the developing myocardium from E13.5. This two-fold higher ratio of ENG protein in veins versus arteries is maintained throughout cardiac development and in the adult heart.ENG is also down-regulated two-fold following mesenchymal transition of endocardial cells to form cardiac valve mesenchyme, whilst expression of the pan-endothelial marker CD31 is completely lost. A subset of epicardial cells (which do not express ENG protein) delaminate and undergo a similar mesenchymal transition to form epicardially derived cells (EPDCs). This transient intra-myocardial mesenchymal cell population expresses low levels of ENG protein, similar to valve mesenchyme.In conclusion, ENG shows dynamic changes of expression in vascular endothelial cells, endocardial cells and mesenchymal cells in the developing heart that vary according to cardiovascular cell type.     

Formation of myocardium after the initial development of the linear heart tube.

Well after formation of the primary linear heart tube, the mesenchymal cardiac septa become largely myocardial, and myocardial sleeves are formed along the caval and pulmonary veins. This second wave of myocardium formation can be envisioned to be the result of recruitment of cardiomyocytes by differentiation from flanking mesenchyme and/or by migration from existing myocardium (myocardialization). As a first step to elucidate the underlying mechanism, we studied in chicken heart development the formation of myocardial cells within intra- and extracardiac mesenchymal structures. We show that the second wave of myocardium formation proceeds in a caudal-to-cranial gradient in vivo. At the venous pole, loosely arranged networks of cardiomyocytes are observed in the dorsal mesocardium from H/H19 onward, in the atrioventricular cushion region from H/H26 onward, and in the proximal outflow tract (conus) from H/H29 onward. The process is completed at H/H stage 43. Subsequently, we determined the potential of the different cardiac compartments to form myocardial networks in a 3D in vitro culture assay. This analysis showed that the competency to form myocardial networks in vitro is a characteristic of the myocardium that is flanked by intra- or extracardiac mesenchyme, i.e., the inflow tract, atrioventricular canal, and outflow tract. These cardiac compartments can be induced to form myocardial networks by a temporally released or secreted signal that is similar throughout the entire heart. Atrial and ventricular compartments are not competent and do not produce the inducer. Moreover, cardiac cushion mesenchyme was found to be able to (trans-)differentiate into cardiomyocytes in the in vitro culture assay. The combined observations suggest that a common mechanism and molecular regulatory pathway underlies the recruitment of mesodermal cells into the cardiogenic lineage during this second wave of myocardium formation through the entire heart.     

Reciprocal interactions between epithelium, mesenchyme, and epidermal growth factor (EGF) in the regulation of mandibular mitotic activity in the embryonic chick

Mandibular epithelia and mesenchyme from chick embryos of Hamburger and Hamilton (H.H.) stage 18-25 were cultured intact, in isolation, or in recombinations in the presence or absence of 5-40 ng/ml epidermal growth factor (EGF). 3H-thymidine labelling demonstrated that mesenchyme influenced epithelial mitotic activity and vice versa. EGF can substitute for the epithelial effect. The stimulation of mesenchymal proliferation by H.H. 18 and 22 epithelia correlated with high levels of epithelial proliferation. Epithelial proliferation was low at H.H. 25 and unaffected by mesenchyme or by EGF. Epithelial stimulation of mesenchymal proliferation began earlier (H.H. 18) than did mesenchymal stimulation of epithelial proliferation (H.H. 22); i.e., within the ages tested, the epithelium initiated these reciprocal mitogenic interactions. That epithelial dependence on mesenchyme coincided with epithelial bone-evoking properties, suggested a) that mesenchyme promotes or maintains epithelial bone-promoting activity and b) that the critical differentiative influence of epithelium on mesenchyme is a mitogenic one. The temporal correlation between a sharp decline in mesenchymal proliferation and termination of the osteogenic epithelial-mesenchymal interaction at H.H. 25 further supports a connection between epithelial maintenance of mesenchymal proliferation and epithelial evocation of osteogenesis.     

Initial expression of type I procollagen in chick cardiac mesenchyme is dependent upon myocardial stimulation

Formation of the atrioventricular (AV) mesenchyme is a critical step in early heart development. Endothelial cells are activated and transformed into a mesenchymal population that invades the cell-free myocardial basement membrane. This process can be duplicated in collagen gel culture, where it has been established that myocardium or its secretory products activate the endothelium. The purpose of the present study was to determine when these activated endothelial and/or mesenchymal cells start producing type I collagen in situ. These results were compared to those obtained from a culture model of mesenchyme formation. The production of type I collagen was monitored using a monoclonal antibody (M38) that recognizes the carboxy-terminal propeptide of human type I procollagen. The initial expression of the latter within activated AV endothelial and mesenchymal cells in ovo was 48 hr following activation. Prior to this time, only the myocardium was reactive with M38. AV explants of early hearts on collagen gels revealed staining of activated endothelial and mesenchymal cells with M38 after 48 hr in coculture with myocardial tissue. Explants that were prevented from activating (myocardium removed) never expressed the M38 antigen. Similarly, AV endothelial monolayers grown in the presence of myocardial conditioned medium activated and expressed type I collagen after 48 hr in culture, whereas those grown in standard medium did not. These results establish the initial expression of type I collagen within activated AV endothelium and mesenchyme. In addition, the data suggest that the expression of type I collagen within the AV mesenchyme may be dependent on extrinsic influences that induce the AV endothelium to transform into mesenchyme.     

Distinct signaling molecules control Hoxa-11 and Hoxa-13 expression in the muscle precursor and mesenchyme of the chick limb bud.

The limb muscles, originating from the ventrolateral portion of the somites, exhibit position-specific morphological development through successive splitting and growth/differentiation of the muscle masses in a region-specific manner by interacting with the limb mesenchyme and the cartilage elements. The molecular mechanisms that provide positional cues to the muscle precursors are still unknown. We have shown that the expression patterns of Hoxa-11 and Hoxa-13 are correlated with muscle patterning of the limb bud (Yamamoto et al., 1998) and demonstrated that muscular Hox genes are activated by signals from the limb mesenchyme. We dissected the regulatory mechanisms directing the unique expression patterns of Hoxa-11 and Hoxa-13 during limb muscle development. HOXA-11 protein was detected in both the myogenic cells and the zeugopodal mesenchymal cells of the limb bud. The earlier expression of HOXA-11 in both the myogenic precursor cells and the mesenchyme was dependent on the apical ectodermal ridge (AER), but later expression was independent of the AER. HOXA-11 expression in both myogenic precursor cells and mesenchyme was induced by fibroblast growth factor (FGF) signal, whereas hepatocyte growth factor/scatter factor (HGF/SF) maintained HOXA-11 expression in the myogenic precursor cells, but not in the mesenchyme. The distribution of HOXA-13 protein expression in the muscle masses was restricted to the posterior region. We found that HOXA-13 expression in the autopodal mesenchyme was dependent on the AER but not on the polarizing region, whereas expression of HOXA-13 in the posterior muscle masses was dependent on the polarizing region but not on the AER. Administration of BMP-2 at the anterior margin of the limb bud induced ectopic HOXA-13 expression in the anterior region of the muscle masses followed by ectopic muscle formation close to the source of exogenous BMP-2. In addition, NOGGIN/CHORDIN, antagonists of BMP-2 and BMP-4, downregulated the expression of HOXA-13 in the posterior region of the muscle masses and inhibited posterior muscle development. These results suggested that HOXA-13 expression in the posterior muscle masses is activated by the posteriorizing signal from the posterior mesenchyme via BMP-2. On the contrary, the expression of HOXA-13 in the autopodal mesenchyme was affected by neither BMP-2 nor NOGGIN/CHORDIN. Thus, mesenchymal HOXA-13 expression was independent of BMP-2 from polarizing region, but was under the control of as yet unidentified signals from the AER. These results showed that expression of Hox genes is regulated differently in the limb muscle precursor and mesenchymal cells.     

Immunofluorescent localization of tenascin during development of the mouse urogenital sinus: Possible involvement in genital duct morphogenesis

Tenascin is a compound of the mesenchymal extracellular matrix and has been proposed as a possible mediator in epithelial-mesenchymal interactions, because of its characteristic distribution in tissues during fetal development. In the present study, we have investigated by immunofluorescence the changes in the distribution of tenascin during development of the mouse urogenital sinus, a process in which tissue interactions were found to be essential. Tenascin first appears in dorsal mesenchyme on days 13-15 of gestation, coinciding with morphological changes of the epithelium. During male development, tenascin accumulates in the dorsal mesenchyme around the junction of Wolffian ducts, but not in the ventral mesenchyme, into which prostatic buds (prostate gland anlagen) project from the sinus epithelium. During female development, the mesenchyme that participates in the downgrowth of the vagina (derived from Müllerian ducts) stains intensively for tenascin. In both of these tenascin-positive areas, the epithelium undergoes conspicuous morphogenetic changes. The results suggest that mesenchymal tenascin could be involved in the epithelial morphogenesis of the sinus, especially in the morphogenesis of the genital ducts.     

Fate map of the dental mesenchyme: dynamic development of the dental papilla and follicle

At the bud stage of tooth development the neural crest derived mesenchyme condenses around the dental epithelium. As the tooth germ develops and proceeds to the cap stage, the epithelial cervical loops grow and appear to wrap around the condensed mesenchyme, enclosing the cells of the forming dental papilla. We have fate mapped the dental mesenchyme, using in vitro tissue culture combined with vital cell labelling and tissue grafting, and show that the dental mesenchyme is a much more dynamic population then previously suggested. At the bud stage the mesenchymal cells adjacent to the tip of the bud form both the dental papilla and dental follicle. At the early cap stage a small population of highly proliferative mesenchymal cells in close proximity to the inner dental epithelium and primary enamel knot provide the major contribution to the dental papilla. These cells are located between the cervical loops, within a region we have called the body of the enamel organ, and proliferate in concert with the epithelium to create the dental papilla. The condensed dental mesenchymal cells that are not located between the body of the enamel organ, and therefore are at a distance from the primary enamel knot, contribute to the dental follicle, and also the apical part of the papilla, where the roots will ultimately develop. Some cells in the presumptive dental papilla at the cap stage contribute to the follicle at the bell stage, indicating that the dental papilla and dental follicle are still not defined populations at this stage. These lineage-tracing experiments highlight the difficulty of targeting the papilla and presumptive odontoblasts at early stages of tooth development. We show that at the cap stage, cells destined to form the follicle are still competent to form dental papilla specific cell types, such as odontoblasts, and produce dentin, if placed in contact with the inner dental epithelium. Cell fate of the dental mesenchyme at this stage is therefore determined by the epithelium.     

The role of the mesenchyme in mouse neural fold elevation. II. Patterns of hyaluronate synthesis and distribution in embryos developing in vitro

Hyaluronate (HA) distribution patterns were examined in the cranial mesenchyme underlying the mesencephalic neural folds of mouse embryos maintained in roller tube culture. Using standard image-processing techniques, the digitized images of Alcian blue-stained or 3H-glucosamine-labeled sections digested with an enzyme specific for HA, were subtracted from adjacent, undigested sections. The resultant difference picture images (DPI) accurately depicted the distribution of stained or labeled HA within the cranial mesenchyme. 3H-glucosamine-labeled HA was distributed uniformly throughout the cranial mesenchyme as 12, 18, and 24 hr of culture. By contrast, the mesenchyme was uniformly stained with Alcian blue at 12 hr, but stain intensity decreased in the central regions of the mesenchyme at 18 and 24 hr. HA distribution patterns were also examined in the cranial mesenchyme of embryos cultured in the presence of diazo-oxo-norleucine (DON), a glutamine analogue that inhibits glycosaminoglycan and glycoprotein synthesis. In DON-treated mesenchyme, Alcian blue staining of HA was decreased from that in controls at 12, 18, and 24 hr. However, incorporation of 3H-glucosamine into HA was increased. The distribution of labeled HA within treated mesenchyme as 12, 18, and 24 hr resembled that in controls at 12 hr. These results indicate that the distribution of HA within the cranial mesenchyme of normal mouse embryos during neural fold elevation and convergence is not determined solely by regional differences in HA synthesis. We propose that HA distribution patterns result from the expansion of the HA-rich extracellular matrix of the central mesenchyme regions. This expansion may play a major role in fold elevation. These results also suggest that DON treatment reversibly inhibits HA synthesis, since treated mesenchymal cells retain the capability of synthesizing HA when provided with a glucosamine substrate. Patterns of 3H-glucosamine incorporation by DON-treated mesenchyme are similar to those observed in control mesenchyme prior to mesenchymal expansion at 12 hr.     

Fibroblast growth factor (FGF)-4 can induce proliferation of cardiac cushion mesenchymal cells during early valve leaflet formation

While much has been learned about how endothelial cells transform to mesenchyme during cardiac cushion formation, there remain fundamental questions about the developmental fate of cushions. In the present work, we focus on the growth and development of cushion mesenchyme. We hypothesize that proliferative expansion and distal elongation of cushion mesenchyme mediated by growth factors are the basis of early valve leaflet formation. As a first step to test this hypothesis, we have localized fibroblast growth factor (FGF)-4 protein in cushion mesenchymal cells at the onset of prevalve leaflet formation in chick embryos (Hamburger and Hamilton stage 20-25). Ligand distribution was correlated with FGF receptor (FGFR) expression. In situ hybridization data indicated that FGFR3 mRNA was confined to the endocardial rim of the atrioventricular (AV) cushion pads, whereas FGFR2 was expressed exclusively in cushion mesenchymal cells. FGFR1 expression was detected in both endocardium and cushion mesenchyme as well as in myocardium. To determine whether the FGF pathways play regulatory roles in cushion mesenchymal cell proliferation and elongation into prevalvular structure, FGF-4 protein was added to the cushion mesenchymal cells explanted from stage 24-25 chick embryos. A significant increase in proliferative ability was strongly suggested in FGF-4-treated mesenchymal cells as judged by the incorporation of 5'-bromodeoxyuridine (BrdU). To determine whether cushion cells responded similarly in vivo, a replication-defective retrovirus encoding FGF-4 with the reporter, bacterial beta-galactosidase was microinjected into stage 18 chick cardiac cushion mesenchyme along the inner curvature where AV and outflow cushions converge. As compared with vector controls, overexpression of FGF-4 clearly induced expansion of cushion mesenchyme toward the lumen. To further test the proliferative effect of FGF-4 in cardiac cushion expansion in vivo (ovo), FGF-4 protein was microinjected into stage 18 chick inner curvature. An assay for BrdU incorporation indicated a significant increase in proliferative ability in FGF-4 microinjected cardiac cushion mesenchyme as compared with BSA-microinjected controls. Together, these results suggest a role of FGF-4 for cardiac valve leaflet formation through proliferative expansion of cushion mesenchyme.     

In vitro studies on the existence of endothelial precursor cells in the subectodermal avascular region of quail wing buds

In vitro studies were performed to investigate the angiogenic capacity of different parts of the avian limb bud. Small pieces of wing mesenchyme of the vascularized core or of the avascular subectodermal region were obtained from quail embryos at stages 18 to 25, and were cultured. The identification of the avascular wing mesenchyme was made possible after injection of India ink via the vitelline vein or by bleeding control during in vivo dissection. Tissue cultures were treated with the QH-1 antibody or/and the endothelial cell marker DiI-Ac-LDL. Endothelial cells were found in cultures of the mesenchymal core and in those of the avascular subectodermal wing mesenchyme. Moreover, their appearance was independent of the stage of the donor embryo. Although there were no vessels, the subectodermal wing mesencyme was able to produce endothelial cells that proliferated and differentiated under in vitro conditions. Thus, endothelial precursor cells probably existed within the avascular wing mesenchyme. These cells might be identical with the QH-1-positive isolated cells that have been described in immunohistochemical studies of this region; they may contribute to the growing capillary plexus of the limb bud.     

Myocardial specificity for initiating endothelial-mesenchymal cell transition in embryonic chick heart correlates with a particulate distribution of fibronectin

The early chick heart tube consists of myocardium and endothelium separated by a myocardially derived basement membrane (MBM). As development proceeds, the endothelium undergoes a transition into mesenchyme in a regionally specific manner; only the atrioventricular (AV) and outflow tract, but not the ventricular endothelium, is transformed into mesenchyme, the progenitor of heart septa and valves. Recent experiments have shown that an EDTA extract of MBM can initiate AV endothelium to form mesenchyme in an in vitro collagen gel culture system. Two-dimensional gel electrophoresis of AV region EDTA extracts showed potentially three isoelectric forms of fibronectin (Fn), while extracts from ventricle contained only two forms. The purpose of the present study was to further investigate the significance of these regional differences by testing of specific myocardial regions (AV vs ventricle) for their ability to induce endothelium to form mesenchyme in vitro, and to immunohistochemically determine if a regionally specific distribution of Fn exists in the MBM that can be correlated with previous electrophoretic data. Embryonic heart regions cultured on three-dimensional collagen gels showed that AV endothelium could only form mesenchyme if cocultured with AV myocardium. Coculture with ventricular myocardial explants did not initiate differentiation of AV endothelium. In contrast, ventricular endothelial cells did not form mesenchyme when cocultured with AV or ventricle myocardium. Immunohistochemical localization of Fn revealed three distinct morphological patterns of distribution in the AV-MBM, i.e., an intense lamina densa staining, diffuse staining in fibrils, and as particles. The Fn localized in particles (0.1 to 0.5 micron in diameter) appeared as a gradient of decreasing concentration extending from the myocardium toward the endothelium. In contrast, no particulate Fn staining was observed in the ventricular region. EDTA extraction selectively depleted the particulate form of Fn. Previous work has shown that this extract, which contains several lower Mr proteins in addition to Fn, is biologically active in initiating mesenchyme formation from AV endothelium in vitro. These results show that a regionally specific interaction of the myocardium with the endothelium is required to initiate the formation of prevalvular mesenchyme. This interaction may be mediated by a multicomponent complex involving Fn and other proteins which appear as a regionally distinct particulate only in areas of endothelial differentiation.     

BMP4 and PTHrP interact to stimulate ductal outgrowth during embryonic mammary development and to inhibit hair follicle induction

The mammary glands develop initially as buds arising from the ventral embryonic epidermis. Recent work has shed light on signaling pathways leading to the patterning and formation of the mammary placodes and buds in mouse embryos. Relatively little is known of the signaling pathways that initiate branching morphogenesis and the formation of the ducts from the embryonic buds. Previous studies have shown that parathyroid hormone-related protein (PTHrP; also known as parathyroid hormone-like peptide, Pthlh) is produced by mammary epithelial cells and acts on surrounding mesenchymal cells to promote their differentiation into a mammary-specific dense mesenchyme. As a result of PTHrP signaling, the mammary mesenchyme supports mammary epithelial cell fate, initiates ductal development and patterns the overlying nipple sheath. In this report, we demonstrate that PTHrP acts, in part, by sensitizing mesenchymal cells to BMP signaling. PTHrP upregulates BMP receptor 1A expression in the mammary mesenchyme, enabling it to respond to BMP4, which is expressed within mesenchymal cells underlying the ventral epidermis during mammary bud formation. We demonstrate that BMP signaling is important for outgrowth of normal mammary buds and that BMP4 can rescue outgrowth of PTHrP(-/-) mammary buds. In addition, the combination of PTHrP and BMP signaling is responsible for upregulating Msx2 gene expression within the mammary mesenchyme, and disruption of the Msx2 gene rescues the induction of hair follicles on the ventral surface of mice overexpressing PTHrP in keratinocytes (K14-PTHrP). Our data suggest that PTHrP signaling sensitizes the mammary mesenchyme to the actions of BMP4, triggering outgrowth of the mammary buds and inducing MSX2 expression, which, in turn, leads to lateral inhibition of hair follicle formation within the developing nipple sheath.     

Immunohistochemical Study of the Origin of the Subepicardial Mesenchyme in the Dogfish (Scyliorhinus canicula)

A mesenchyme appears at the subepicardium of the vertebrate embryo closely following the epicardial ensheathing of the heart. We have suggested earlier that a localized epithelial-mesenchymal transition (= transdifferentialion) of the epicardial mesothelium might explain the origin of the subepicardial mesenchymal cells (SEMC) in a primitive vertebrate, the dogfish Scyliorhinus canicula. We have tested our hypothesis through the immunolocalization, in the embryonic dogfish heart, of three proteins involved in the epithelial—mesenchymal transitions. Fibronectin immunoreactivity (FN-IR) was present between epicardium and myocardium from the earliest stages of the epicardial development. However, in embryos ranging between 20 and 35 mm TL, FN-IR disappeared from the basal surface of the epicardial cells at the atrioventricular and conoventricular grooves and increased in association with the SEMC. Proliferating-cell nuclear antigen immunoreactivity was intense in most epicardial cells and SEMC in these areas and developmental stages, but it declined in later embryos. The JB3 antigen, a fibrillin-related protein, was delected in the cytoplasm and basal surface of the epicardial cells, as well as on the SEMC surface. These immunohistochemical patterns were remarkably similar at the subepicardium and at the endocardial cushions, which are populated by mesenchymal cells derived from the transdifferentiation of the endocardium. © 1998 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd. All rights reserved     

Analysis of the endocardium and cardiac jelly in truncal development in the cardiac lethal mutant axolotl Ambystoma mexicanum

Recessive mutant gene c in axolotls results in a failure of the heart to function because of abnormal embryonic induction processes. The myocardium in this mutant lacks organized sarcomeric myofibrils. The present study was undertaken to determine if developmental abnormalities were evident in other areas of the heart besides the myocardium. A detailed comparative survey of the structure of developing normal and mutant hearts, including the endocardium, its cellular derivatives, and the extracellular matrix, known as cardiac jelly, showed that in the mutant there are fewer than the normal number of endocardial cells lining the heart lumen, the number of mesenchyme cells is reduced, and the cardiac jelly area is greatly enlarged in the posterior part of the truncus adjacent to the ventricle.     

An instructive role for the interstitial matrix in tissue patterning: tissue segregation and intercellular invasion

Intercellular invasion is the intrusion of the cells of one tissue into space occupied by a second tissue. The alternative situation to invasion, one characteristic of most coherent tissues, is segregation, with identifiable boundaries existing between contiguous tissues. The interfaces between mesenchymal and myocardial tissues in the developing avian heart show a profoundly different character in different regions of the heart: the interface between epicardial mesenchyme and heart wall myocardium is planar, without intermingling of the two cell types, whereas the interface between endocardial cushion mesenchyme and myocardium is diffuse, with extensive invasion of both tissue types across the border to produce intermingling of the two tissues. Thus, invasion and tissue segregation coexist in different regions of the mesenchyme-myocardium contact zone. Investigation of the involvement of the interstitial matrix in invasion and segregation has been conducted by maintaining the two tissues in mutual contact in organ culture. Investigation of the mechanisms by which the two cell types sort out in randomized chimeric tissue reaggregates has provided insight into the conditions for tissue segregation. We have modeled invasion in organ culture by fusing aggregates of myocardial cells with aggregates of cardiac mesenchymal cells. Cells of both tissues invaded the partner aggregate during a period of 1-3 d of coculture. Both invasion and segregation in the aggregates appear to depend on the presence or absence of a fibronectin-rich interstitial matrix elaborated by the cardiac mesenchyme. During sorting, the matrix appears selectively in regions occupied by the mesenchyme. Under conditions of culture that are nonpermissive for matrix deposition, sorting fails to occur. Stimulation of matrix deposition by addition of serum, transforming growth factor beta, or isolated matrix itself is accompanied by sorting out of the two tissues. Sorting out is blocked reversibly by inclusion of the fibronectin adhesion site peptide, GRGDSP. Invasion of fused aggregates is preceded by a redistribution of the fibronectin-containing matrix of the mesenchymal aggregate such that matrix-poor regions come to occupy the interface with the myocardial partner aggregate. The invasion that ensues involves mesenchymal cells emigrating from, and myocardial cells intruding into, matrix-poor regions of the mesenchymal aggregate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney

During kidney development and in response to inductive signals, the metanephric mesenchyme aggregates, becomes polarized, and generates much of the epithelia of the nephron. As such, the metanephric mesenchyme is a renal progenitor cell population that must be replenished as epithelial derivatives are continuously generated. The molecular mechanisms that maintain the undifferentiated state of the metanephric mesenchymal precursor cells have not yet been identified. In this paper, we report that functional inactivation of the homeobox gene Six2 results in premature and ectopic differentiation of mesenchymal cells into epithelia and depletion of the progenitor cell population within the metanephric mesenchyme. Failure to renew the mesenchymal cells results in severe renal hypoplasia. Gain of Six2 function in cortical metanephric mesenchymal cells was sufficient to prevent their epithelial differentiation in an organ culture assay. We propose that in the developing kidney, Six2 activity is required for maintaining the mesenchymal progenitor population in an undifferentiated state by opposing the inductive signals emanating from the ureteric bud.     

Expression of nuclear estrogen-binding sites within developing human fetal vagina and urogenital sinus

An autoradiographic study of nuclear estrogen binding was performed in developing human urogenital sinuses and vaginas derived from first and second trimester specimens. Nuclear estrogen binding was detected in all specimens greater than or equal to 10 weeks of gestation within mesenchymal cells. Nuclear labelling within epithelium was observed only in those specimens whose development and differentiation was advanced. Thus, mesenchyme appears to be the initial estrogen target tissue within the developing human vagina and may play a fundamental role in estrogen-induced teratogenesis of the human genital tract.